The present invention generally relates to methods utilizing optical systems for the detection of mycotoxins in grain. More specifically, the invention is directed to the detection of the mycotoxin aflatoxin in pre and post-harvest corn samples. Furthermore, the invention can also be deployed in the field where, due to various environmental changes, aflatoxin contamination naturally occurs.
Aflatoxins are toxic secondary metabolites produced by fungi of the genus Aspergillus under stressed conditions. Aflatoxin producing members of Aspergillus are common and widespread in nature. They can colonize and contaminate grain before harvest or during storage. Host crops are particularly susceptible to infection by Aspergillus and consequent aflatoxin contamination, following prolonged exposure to a high humidity environment or damage from stressful conditions such as drought. Aflatoxins have received greater attention than any other mycotoxins because of their demonstrated potent carcinogenic effect in susceptible laboratory animals and their acute toxicological effects in humans. Because absolute safety can never be realistically achieved, many countries have attempted to control exposure to aflatoxins by imposing regulatory limits on commodities intended for use as food and feed.
Sampling and sample preparation remain a considerable source of error in the analytical identification of aflatoxins. Thus, systematic approaches to sampling, sample preparation, and analysis are absolutely necessary to determine aflatoxins at the parts-per-billion level. A common feature of all sampling plans is that the entire primary sample must be ground and mixed so that the analytical test portion has the same concentration of toxin as the original sample. The unfortunate result of this type of aflatoxin determination is the destruction of the original sample.
All analytical procedures include three steps: extraction, purification, and determination. Solid-phase extraction is used to clean up test extracts before instrumental analysis (for example, thin layer or liquid chromatography) to remove co-extracted materials that may interfere with the determination of target analytes.
Thin layer chromatography (TLC), also known as flat bed chromatography or planar chromatography, is one of the most widely used separation techniques in aflatoxin analysis. Thin layer chromatography has been considered the official method and the method of choice of the Association of Official Analytical Chemists (AOAC) to identify and quantify aflatoxins at levels as low as 1 ng/g. The method is also used to verify findings by newer, more rapid techniques.
Liquid chromatography (LC) is similar to TLC in many respects, including analyte application, stationary phase, and mobile phase. Liquid chromatography and TLC complement each other. It is quite common for an analyst to use TLC for preliminary work to optimize LC separation conditions. Liquid chromatography methods for the determination of aflatoxins in foods include normal-phase LC (NPLC), reversed-phase LC (RPLC) with pre- or before-column derivatization (BCD), RPLC followed by postcolumn derivatization (PCD), and RPLC with electrochemical detection.
Thin layer chromatography and LC methods for determining aflatoxins in food are laborious and time consuming. Often, these techniques require knowledge and experience of chromatographic techniques to solve separation and interference problems. Through advances in biotechnology, highly specific antibody-based tests are now commercially available that can identify and measure aflatoxins in food in less than 10 minutes. These tests are based on the affinities of the monoclonal or polyclonal antibodies for aflatoxins. The three types of immunochemical methods are radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and immunoaffinity column assay (ICA) similar to U.S. Pat. No. 4,818,687.
Safety is a key issue for scientists working in the aflatoxin area. Steps must be taken to minimize exposure to the toxins as well as to the producing microorganisms, Aspergillus flavus and Aspergillus parasiticus. A safety program should be established that meets the requirements of the Laboratory Standard of the Occupational Safety and Health Administration (1990) and the guidelines of the National Institutes of Health (1981) covering use of chemical carcinogens.
A non-invasive method for separating aflatoxin-contaminated from non-contaminated grains, kernels, seeds and nuts was disclosed in U.S. Pat. No. 4,795,651 to Henderson, et al., in 1989. The process involved using dynamic flotation to separate contaminated and uncontaminated substances based on specific gravity because aflatoxin contaminated commodities appear to have lower specific gravity than uncontaminated ones. Although quite reliable, the process is lengthy and not practical for processing large quantities of grain as it requires wetting and re-drying individual grains including corn kernels.
U.S. Pat. No. 4,535,248 discloses an approach for non-invasive rapid detection of aflatoxin. This patent utilizes long wave ultraviolet radiation for detecting aflatoxin contamination in almonds. The method detects the presence of aflatoxin based on evidence of violet-purple fluorescence. A similar method utilizing bright greenish-yellow (BGY) fluorescence was employed for detecting possible aflatoxin contamination in corn kernels. The reasoning behind using the method was based on the premise that seed contaminated with A. flavus is often associated with BGY fluorescence. However, the presence of BGY fluorescence may or may not indicate aflatoxin contamination in corn kernels, and thus introduces high false positive errors in detection. Therefore, the method is no longer used in corn as an aflatoxin detection method, but only a screening method for kernels requiring further investigation.
Hyperspectral imaging systems have been used for a diverse range of remote sensing and other analytical techniques, such as is disclosed, for example, in U.S. Pat. No. 5,790,188 and the related U.S. Pat. No. 6,211,906. Hyperspectral imaging has also been used in conjunction with microscopic optical systems, such as disclosed, for example, in U.S. Pat. No. 6,495,818. In such systems, radiation reflected by or emanating from a target or specimen is detected in a large number of narrow contiguous spectral bands, producing a data set which is distributed not only spatially, but spectrally as well. That is, for each pixel within an image of the target, information is recorded in each of the spectral bands, thereby producing a three-dimensional hyperspectral image cube, in which spectral information for each pixel is distributed across a spectral axis perpendicular to the spatial axes.
Narrow band spectral reflectance across a wide spectral range (for example, UV, visible near-infrared, and short wave near-infrared) can provide rich spectral signature information regarding suspicious targets. The spectral signature can then be used for separation of specific targets such as aflatoxin contaminated and non-contaminated corn. The process involves irradiating corn kernels with magnetic radiation (such as a light source working under a certain wavelength range), measuring reflected radiation from the kernels with electronic devices (such as a CCD array that is sensitive in a certain wavelength range), composing a target signature from the captured signals, and implementing detection/separation algorithms for detecting aflatoxin in corn. The present invention applies this spectral based approach along with a UV light source and uses spectral fluorescence information for aflatoxin detection.
Similar systems that use spectral information for various applications have been described in the past. Most such systems utilize a technique in which the suspicious item is irradiated with light having a frequency (for example, UV, visible near-infrared, and short wave near-infrared) such that it causes the emission of fluorescent radiation upon striking the target. The fluorescent light from the target is then measured and compared with a threshold value. If the light thus gathered exceeds the threshold, the detection algorithm can generate a signal indicating the presence of the target. Such a system is disclosed, for example in U.S. Pat. No. 4,622,469 to Akiyama for detecting rotten albumen in broken raw eggs, U.S. Pat. No. 6,512,236 B2 to Seville for viewing patterns of fluorescently stained DNA, protein or other biological materials, and U.S. Pat. No. 5,914,247 to Casey et al., for a fecal and ingesta contamination detection system based on the premise that the emission of fluorescence between 660 and 680 nm is indicative of the presence of ingesta or fecal matter.
Radiation-induced fluorescence has also been applied in vegetation remote sensing studies. For example, it has been shown that there is a shift in the fluorescence signature of a stressed plant compared to a healthy plant (Barbini, et al., Laser Remote Monitoring of the Plant Photosynthetic Activity, SPIE, Vol. 2585, (1995), pp. 57-65).
Fluorescence hyperspectral images were collected with an aggregation of corn kernels (Yao et al., Hyperspectral Bright Greenish-Yellow Fluorescence (BGYF) Imaging of Aflatoxin Contaminated Corn Kernels, Proc. of SPIE, Optics for Natural Resources, Agriculture, and Foods, 2006). The study reported that the aggregated bright greenish-yellow (BGY) fluorescence corn kernels had fluorescence peaks at around 500 nm and 514 nm. It was later determined that the imaging system was not properly calibrated for wavelength thus the reported fluorescence spectra were not correct. The experiment used 365 nm UV-LED as excitation light source. However, in this initial exploratory study, no excitation and emission filters were used. The recorded spectra were mixed with signal from the light source. As a consequence, the resulted spectra were not representative to aflatoxin contaminated corn kernels. Lastly, the study used only one group of BGY fluorescence corn kernels for imaging. In order to document the relationship between fluorescence spectra and aflatoxin contamination, single kernel imaging and chemical analysis is needed. Consequently, no conclusion could be drawn based on this study.
Two-band fluorescence image data at 450 nm and 505 nm were used for aflatoxin detection in corn kernels (Ononye et al., Automatic Detection of Aflatoxin Contaminated Corn Kernels Using Dual-Band Imagery, Proc. of SPIE, Vol. 7315, 73150R, 2009). The corn sample size was 25 g. The samples were excited with 365 nm UV light source and image data were collected with a Nuance Multispectral Imaging System (Cambridge Research Institute, MA). The imaging system was equipped with a built-in liquid crystal tunable filter that was tuned to 450 nm and 505 nm. Since the study used 25 g of corn kernels as one sample, the relationship between fluorescence spectra and aflatoxin contamination in single corn kernel could not be established. This study did not produce a plot of the fluorescence spectra. Thus, characteristics of the fluorescence spectra, such as fluorescence peak and shift could not be determined in the study.
The present invention provides techniques and devices for detection of aflatoxin in corn by fluorescence spectral imaging. The fluorescence spectral image is a three dimensional “image” where one dimension contains spectral information and the other two dimensions contain spatial information. The spectral data of the image can be analyzed on a pixel-by-pixel basis for the mycotoxin in question and its spatial extent. Thus, the techniques are non-invasive and do not require introduction of agents typically required to facilitate interaction with illumination sources. The techniques also have minimal requirements in sample preparation and can generate identification results in a short period of time once the image is acquired.
An objective of the present invention is to use spectral information of the mycotoxin aflatoxin in order to detect it in a target material, such as in corn samples. The invention may be embodied in the form of, for example, tabletop lab equipment or a portable system to be used at specific inspection sites. Our research has discovered that a single aflatoxin-contaminated corn kernel has distinct spectral fluorescence features. Our research further shows that these spectral fluorescence features can be used for aflatoxin contamination detection and quantification. The invention thus incorporates technical details and possible embodiments for aflatoxin detection in corn using spectral fluorescence. This invention in real time will allow the user to collect an infinite number of images and requires no knowledge of aflatoxin chemistry.
Another objective of the present invention is to provide for implementation of fluorescence excitation sources, data capture devices, database of reference fluorescence spectra, processing methods, and identification algorithms.
A further objective of this invention is to provide a method for processing the fluorescence spectra emitted from corn kernels, a method which also produces a signal that reliably indicates aflatoxin contamination, and apparatus which implement such method.
Another objective of this invention is to differentiate between healthy and aflatoxin contaminated corn based on fluorescence emission under direct long wavelength ultraviolet excitation, and utilize peak fluorescence and peak fluorescence shift for aflatoxin detection in corn.
Another objective is to provide details of a tabletop system, which is a possible embodiment of this invention that can be used in lab environments such as analytical labs, for aflatoxin detection in corn. The tabletop system is for detecting contamination in larger corn samples (approximately 25-1000 g). Such a system includes an operation computer, fluorescence excitation source with a specific wavelength centered around 365 nm, image capture devices, database of reference fluorescence spectra, processing methods, and identification algorithms.
An additional objective is to provide details of a portable system which can be used at various inspection sites such as grain inspection stations, silos, or in the field for aflatoxin detection in corn. The portable system is for detecting contamination in smaller corn samples (approximately from a single corn kernel to an entire corn ear). Such a system includes an embedded operation computer, fluorescence excitation source with a specific wavelength centered around 365 nm, spectral data capture devices, database of reference fluorescence spectra, processing methods, and identification algorithms.
A further objective is to incorporate a fluorescence excitation source in the detection system. Such excitation source can consist of a UV fluorescence lamp or a UV LED that radiates at long ultraviolet wavelengths and an excitation filter centered at 365 nm.
An additional objective of this invention is to provide a non-invasive aflatoxin detection system that can quickly and accurately detect and quantify aflatoxin contamination in corn.
The invention includes an imaging camera, micro-spectrometer array, or photodiode detector array for fluorescence spectral data acquisition, a fluorescence excitation source, digital reference library of pertinent fluorescence spectra, and the appropriate identification algorithm/software. The identification process is completely non-invasive, and rapid for real time work. The procedure can be automated, and its determination is objective because no human judgment is involved.
Other objects, advantages and novel features of the present invention will become apparent from the following detailed description of the invention when considered in conjunction with the accompanying drawings.